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Lecture 21

Tissue Engineering

Tissue Engineering: a field that seeks to replace, repair or enhance biological function at the scale of a tissue or organ by manipulating cells via their extra-cellular environment

Objectives:

1. Fulfill a biomechanical role (bone, cartilage) 2. Replace physiological function (liver, nerve) 3. Deliver secretory products (insulin) 4. A combination of the above

3 Main Approaches:

1. Extracorpeal/cell encapsulation (3) 2. In vitro synthesis (1-4) 3. In vivo synthesis (1-4)

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1. Extracorpeal/Cell Encapsulation

Method:

1. Encapsulate cells of interest in semipermeable membrane 2. Implant encapsulated device or connect ex vivo

3. Cells secrete product � therapy 4. Remove/disconnect device when therapy concluded

O2, nutrients

CO2, waste

Cells suspended in gel

T cells, B cells

Semipermeable membrane

Immune system: Ab,

Therapeutic molecules

Advantages: ¾�Natural theraputic response from living cells ¾�Use of nonhost cells—immunoisolation

Issues:

¾�Potential for undesirable immune response from adsorption of complement proteins (similar to blood filtration membranes)

¾�Potential for thrombosis formation (Anti-coagulants used during ex vivo treatment)

¾�Potential for rupture of implanted devices

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Applications Investigated:

¾�Diabetes treatment* ¾�Chronic pain* ¾�Neurodegenerative diseases: ALS (Lou Gehrig’s disease, neuromuscular),

Parkinson’s, Alzheimer’s, Huntington’s disease (progressive brain death) ¾�Dwarfism ¾�Anemia/Hemophelia ¾�Macular degeneration (blindness) ¾�Cancer ¾�Liver Failure* * = clinical trials

Device Examples

Encapsulated Islets (Islet Technology Inc., St Paul, MN) CapCell: implantable membrane-encapsulated islets for glucose regulation

Cells: insulin-producing islets Use: long-term treatment of diabetes Device: alginate-based membrane confines islets Treatment: islets transplanted into patient’s pancreas; patients’ blood flows thru membrane; islets detect glucose level variations & respond through insulin production Status: preclinical trials (islet transplantation in clinical trials)

Islet cells

Alginate-based membranes

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Arbios Systems, Inc. (recently acquired from Circe Biomedical)

HepatAssist System: an extracorporeal, bioartificial liver support system

Cells: primary porcine hepatocytes (pig liver cells) Use: temporary liver function for transplant candidates Device: hollow fiber bioreactor, oxygenator, pump Treatment: plasma circulated through bioreactor and

recombined with blood cells Status: Phase I trials completed

Image removed due to copyright considerations.

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2. In vitro Synthesis

Method:

1. Cells seeded in vitro on scaffold device 2. Cells maintained in culture to expand population &

develop tissue organization (in static culture or bioreactors)3. Device implanted once cell colony is established 4. Device degrades, scaffold replaced by remodeled tissue

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Advantages: ¾�Natural theraputic response from living tissues ¾�Permanent therapy ¾�Allows control and quantification not easily obtained in vivo

Issues:

¾�Cell sources - possibility of rejection - tumorogenicity—cell lines

¾�Full organ restoration challenges (e.g., skin)

Applications Investigated:

¾�Vasculature (resorbable & nonresorbable) ¾�Liver tissue ¾�Nerve tissue ¾�Cartilage* ¾�Cornea* ¾�Bladder* ¾�Skin* ¾�Bone ¾�Ligament ¾�Tendon ¾�Muscle ¾�Heart valve ¾�Heart

Company Updates:

Advanced Tissue Sciences, Inc., La Jolla. CA: filed for bankruptcy under Chapter 11, Oct. ’02

Organogenesis, Canton, MA: filed for bankruptcy under Chapter 11, Sept. ’02; Apligraf artificial skin technology acquired by Novartis

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3. In vivo synthesis

Method:

1. Implant porous scaffold device2. Cellular ingrowth in vivo

3. Scaffold replaced by remodeled tissue

Advantages: ¾�Natural theraputic response from living tissues ¾�Permanent therapy ¾�No cell source problems

Issues:

¾�Uncontrolled biological response to implanted scaffold

Applications Investigated:

¾�Vasculature ¾�Skin* ¾�Bone* ¾�Nerve ¾�Ligament ¾�Cartilage (Knee Meniscus)

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Scaffolds for Tissue Generation

Purpose: replace functions of extracellular matrix (ECM)

ECM functions:

1. cell anchorage 2. cell orientation 3. cell growth 4. mechanical integrity to neo-tissue 5. tissue microenvironment 6. cell differentiation 7. sequester, store & present soluble regulatory proteins 8. blueprint for tissue organization (e.g., biomineralized tissue)

ECM types:

Basal Lamina (basement membrane): directly underlying epithelial cells; contains laminin, collagen, fibronectin, vitronectin

Stromal tissue (interstitial matrix): provides structural integrity; contains matrix-secreting cells (fibroblasts, osteoblasts), collagen, elastin, fibrillin, fibronectin, vitronectin, GAGs, glycoproteins, regulatory proteins

Basal lamina

Stromal tissue

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Resorbable Tissue Engineering Scaffolds:

1. Collagen-matrix e.g., artificial skin drawback: immunogenic

2. Biodegradable polymers: PLA, PGA, PLGA e.g., cartilage

drawback: no adhesion sites (can build in RGD)

3. Hydroxyapatite, Bioglass e.g., bone regeneration

drawback: brittle, low strength

Processing of Tissue Engineering Devices

A. Design Issues

1. Cell density must be sufficiently high to enable tissue formation, deliver therapy

2. Transport of nutrients/oxygen/waste nutrients must reach cells within the scaffold/encapsulation device

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Blood vessel Scaffold/encapsulation device

Co

Cnut

x

Limiting distance from nutrients can be gauged from the Thiele ratio, S

(dimensionless ratio of consumption to supply)

k x2 D = nutrient diffusivity in device (cm2/sec)U Co = nutrient concentration at source (mol/cm3)S DC k = cell nutrient uptake rate constant (mol/sec/cell)

o x = distance from nutrient source (bloodstream) (cm) U = cell density in device (cells/cm3)

S>>1 � cells consume more than can be delivered S<<1 � supply greater than demand S = 1 � supply balances demand; use as limit estimate for device design

A rule of thumb in designing tissue engineering devices: xmax= 500 Pm.

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3. Mechanical support - Critical problem for hard tissue scaffolds

- Influenced by¾�materials choices ¾�processing (orientation of polymers & composites)

4. Tissue organization blueprint

Cell migration guidance chemical & morphology effects (chemo/hapto/durotaxis)

Cell Patterning microcontact printing, microlithography

Spatial Organization of Muliple Cell Types - most organs of more than one cell type - pattern based on different ligands, ligand densities, ligand affinities

B. Scaffold Fabrication

Objective: Continuous, high-surface area scaffolds

1. Fabrics

¾�Woven/nonwoven fibers ¾�Mechanical interlocking � pliable, 3D matrix ¾�Porosity and pore size roughly controlled

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2. Bonded fibers

¾�PGA fibers dipped in PLLA/CH2Cl2 solution ¾�Heat treat fibers at Tg,PGA < T < Tm,PLLA to bond PGA to PGA ¾�Dissolve away PLLA ¾�Improved mechanical properties over fabrics; similar porosity

3. Freeze-dried Foams

¾�Polymer solution immersed in liquid N2 � phase separation ¾�Frozen solvent sublimates leaving porous scaffold ¾�Pore size ~ O of spinodal decomposition � controlled pore structure

4. Salt-leached Foams

¾�polymer solution mixed with uniform salt crystals ¾�Solvent evaporates leaving solid polymer/salt composite

Immerse in H2O to leach out salt¾�¾�Controlled porosities up to 93% (< 2 mm thick)

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2. Encapsulation Membranes

¾�Cast concentrated solution onto substrate (flat or tubular)¾�Substrate immersed into a nonsolvent bath ¾�Coagulation of asymmetric membrane results

Microporous substructure (mechanical rigidity, high flux)

Dense surface layer (bath side) (semi-permeable)

¾�Cells suspended in gel within sealed membrane tube (length ~ 3 cm) ordisk (dia ~ 2-3 cm)

Gel & cells

~3 cm

Semipermeablemembrane

600-1200 Pm

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¾�Membrane characteristics

- Molecular weight cutoff: typically ~30-70 kg/mol (<100 nm dia. pores)

Note: Ab ~150 kg/mol

Molecule/Moiety Size

O2, H2O, salts 2-3 �

Lipids, glucose 10 Å

Serum proteins, endotoxins 100 Å

Viruses 1000 Å

Bacteria 104 Å

White blood cells, platelets 105 Å

- Matrix examples: polysaccharides, alginate/chitosan coacervate, collagen

- Body: PAN-PVC, PP, polycarbonate, cellulose nitrate, acrylic

- Shape & Size: disks vs. tubes

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Disks Tubes

Mass transport Favored diameter restrictions

Susceptibility to high surface area favored clotting increases clot propensity

Cell # 50-100M 5M

Cell # required

Diabetes 109

Clotting factor 107-108

CNS therapies 106-107

D. Current Challenges

1. Micromechanical effectsCell differentiation and growth (especially in load-bearing tissues) can be affected by micromechanical stresses transmitted by the scaffold

2. Cell function deterioration

3. Cross-application to other areas (gene therapy, drug delivery)

4. Multicellular tissues and organs- Complex, multicomponent structures (vascularized tissues)- Regeneration-inducing factors (proteins) only known for blood & bone

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Basic Tissue Cell Types and Functions

Cell type Tissue Function Example

epithelial covers external (ex, skin) & internal (ex, intestine, blood vessel) organ surfaces

endothelial cells

connective houses nerves & blood vessels generation),

cartilage, bone

nerve peripheral nerve

supports other body tissues; fibroblasts (ECM

muscle specialized for contraction; smooth, skeletal, cardiac

generate electrical signals & secrete neurotransmitters

brain cells,

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Cell Regeneration Capability

Category Normal Response to Examples

replic. rate injury

renewing/ High; modest n skin, intenstinal labile via stem cell mucosa, bone

differentiation marrow

Expanding/ Low large n endothelium, stable fibroblasts,

hepatocytes, osteoblasts

Static/ None No replication; heart muscle permanent replaced by scar cells, nerve cells

tissue